Laser induced fluorescence capillary interface

ABSTRACT

An optical scheme that substantially eliminates spherical aberration and coma, thereby substantially improving fluorescence excitation and collection efficiency. The optical scheme utilizes a laser beam focused by an optical component through the curved surface of a hyper-hemisphere. The hyper-hemisphere focuses the beam sharply at a known point while avoiding spherical aberration and coma. The optical scheme includes both a hyper-hemisphere and a hemisphere. Both the hyper-hemisphere and hemisphere have a substantially planar surface. The substantially planar surface of the hyper-hemisphere is optimally located so that a capillary or cell can be positioned at an internal aplanatic radius. This results in an aplanatic focus at the capillary or cell such that the spherical aberration and coma are zero. A single hyper-hemisphere having a substantially planar surface can be used, wherein the capillary is located at an aplanatic point on the substantially planar surface of the single hyper-hemisphere.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of the U.S. patent application Ser.No. 09/379,936 filed Aug. 24, 1999 now U.S. Pat. No. 6,239,871 which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of spectroscopy, and moreparticularly to spectroscopy of samples occupying small volumes.

BACKGROUND OF THE INVENTION

Capillary electrophoresis (CE) is a separation technique based on thedifferential migration of charged particles in an electric field. A thincapillary (20-100 μm internal diameter) is filled with an electrolyteproviding a medium in which analytes can migrate through. The sample isintroduced at one end of the CE unit. An electric field typically of100-600 V cm⁻¹ is applied across the capillary facilitating analytespecies migration according to their electrophoretic mobility (u)passing a detector as they migrate (usually UV or fluorescence) at ornear the end of the capillary.

This separation technique as well as others, such as packed capillaryliquid chromatography, capillary electrochromatography and supercritical chromatography, require spectroscopic measurements to be madeon extremely small volumes of flowing liquid samples. The typicalapplication has a sample flowing through a fused silica capillary tubewhere inside diameters range from 15 to 150 micrometers and the outsidediameters range from 150 to 300 micrometers. Various techniquespresently are used for directing light from a suitable source intoand/or through such a small volume sample cell, as well as taking thelight emanating from the inside of the cell and directing it toward alight detecting or analyzing instrument to effect optical analysis ordetection of samples contained in the cell. Alignment of the opticalsystem to efficiently direct the light from the source to the capillarycell, particularly to the bore and sample therein, and/or to direct theradiation emanating from the cell to a detector, presents problems.

The underlying problem is generally related to selecting components andprecisely aligning them for the purpose of directing light from a lightsource such as a laser to, or through, to a volume of interest which hasa small cross-sectional area perpendicular to the optical access.Similar problems are associated with collecting the light that emanatesfrom a volume of interest and directing it to a photodetector oranalyzer.

An implementation described in U.S. Pat. No. 5,037,199 to Hlousekutilizes an optical scheme which attempts to solve these focusing andalignment problems. In Hlousek's disclosure, a laser beam is focusedinto the lumen of a separations capillary or cell using a ball lens. Theball lens and the capillary/cell are mounted together as a unit. A lensfocuses light from a source onto the ball lens. The size and shape ofthe light may be controlled and/or selected by placement of one or moresuitably shaped apertures on axis with the source and thecapillary/cell. The sphere or ball lens concentrates light by acting asa very short focal length lens to convert slowly converging light fromthe laser beam source to a rapidly converging cone of light that willimage the source into or through the volume of interest in the cell.

A ball lens used this way suffers severe aberrations, in particular,spherical aberration and coma, making the laser focus larger thandesirable. Refraction of light rays at the cylindrical outside surfaceof the capillary causes astigmatism and further enlargement of the focalspot. In addition, light is lost due to surface reflections at theball-lens-to-air and air-to-capillary interfaces. Consequently,efficiency of the fluorescence excitation of the sample suffers.Similarly, the ability to efficiently collect the beam is negativelyimpacted.

SUMMARY OF THE INVENTION

The present invention provides an optical scheme that substantiallyeliminates spherical aberration and coma, thereby substantiallyimproving collection efficiency and fluorescence rib excitation.

According to the invention, the optical scheme utilizes a laser beamfocused by an optical component, such as a microscope objective, throughthe curved surface of a hyper-hemisphere. The hyper-hemisphere focusesthe beam sharply at a known point while avoiding spherical aberrationand coma.

In one embodiment, the optical scheme comprises a hyper-hemisphere and ahemisphere. Both the hyper-hemisphere and the hemisphere have asubstantially planar surface. The substantially planar surface of thehyper-hemisphere is optimally located at an internal aplanatic radiuswhere a capillary or cell can be positioned. This results in anaplanatic focus at the location of the capillary lumen whereat thespherical aberration and coma are zero. An aluminized hemisphericalexterior surface of the hemisphere retro-reflects the beam, therebyproviding a second pass through the sample volume.

In one implementation, each of the substantially planar surfaces of thehyper-hemisphere and the hemisphere has a groove disposed therein. Thehyper-hemisphere and the hemisphere are mated by placing in contacttheir substantially planar surfaces. Upon mating, the grooves in thehyper-hemisphere and the hemisphere form a channel in which a fusedsilica capillary is placed. The lumen of the fused silica capillary isthereby located at a second aplanatic point of the hyper-hemisphere, aswell as at the center of the retro-reflecting hemisphere. Fluorescentlight emitted by the small excited volume of interest is collected usingthe same optics, and directed to a detection means using a dichroicbeamsplitter by means well known to those skilled in the art. Theairspace between the fused silica capillary and the groove is filledwith an index-matched gel or liquid, so that the hyper-hemisphere,capillary and hemisphere become a single optical element, therebyeliminating any reflections or losses at the air/silica interface, andalso avoiding astigmatism due to the cylindrical capillary boundary.

Alternatively, no groove is formed in the substantially planar surfaces.Instead, each of the substantially planar surfaces can be ground backsuch that the fused silica capillary fits between the hyper-hemisphereand the hemisphere maintaining the optical scheme, i.e., facilitatingpositioning of the capillary adjacent to the planar surface(s) at apoint of aplanatic focus. The air-space between the hyper-hemisphere andthe hemisphere on either side of the capillary is filled withindex-matching substance, i.e., gel or liquid. The gel or liquid isindex-matched at least to the capillary.

In another embodiment, according to the invention, only a singlehyper-hemisphere having a substantially planar surface is used. Thecapillary is located at an aplanatic point on the singlehyper-hemisphere's substantially planar surface.

Features of the invention include provisions of a spectroscopy system,wherein the precise location of which is relatively insensitive tomovement of the hyper-hemisphere/capillary assembly. The systempossesses a high tolerance for focusing errors and is implemented as asimple assembly.

The hyper-hemisphere used in the present invention increases thenumerical aperture (N.A.) of the system by approximately 50%, therebyincreasing the collection efficiency. Further, by effectively formingone optical component with the capillary, it eliminates reflectionlosses as well as the lensing effect of the outer capillary wall.Additionally, the single hyper-hemisphere approach gives the system avery high tolerance for alignment errors.

BRIEF DESCRIPTION OF THE DRAWING

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments, taken in conjunction with the accompanyingdrawing in which:

FIG. 1 is a diagram illustrating the optical principles underlyingaplanatic capillary interface(s) according to the invention;

FIG. 2(a) is a diagram of an aplanatic capillary interface according toa first embodiment of the invention;

FIG. 2(b) illustrates a variation of the first embodiment of theinvention in which no grooves are formed in planar surfaces;

FIG. 3 is a diagram of an aplanatic capillary interface according toanother embodiment of the invention; and

FIG. 4 illustrates an optical system including an aplanatic capillaryinterface according to the invention.

DETAILED DESCRIPTION

The present invention comprises an optical apparatus designed to allowfor spectroscopic measurements of extremely small samples whilemaintaining a high tolerance for alignment and focusing errors.

Generally, in various embodiments according to the invention, the mainoptical component is a hyper-hemisphere with a substantially planarsurface mated to a hemisphere comprising a substantially planar surface.FIG. 1 illustrates a spherical lens of refractive index “n,” and radiusof curvature “R.” If light is focused so as to form a virtual object ata distance nR from the spherical surface's center of curvature, then thelight is brought to a real focus at a distance R/n, a point inside thespherical lens. This focus is aplanatic, i.e., the spherical aberrationand coma are zero. The distance from the center of the lens to theaplanatic point is called the internal aplanatic radius. In the presentinvention, the spherical lens is ground into a hyper-hemisphere with thesubstantially planar surface located at the internal aplanatic radius.In this way, light can be focused aplanatically on a capillary placedagainst the substantially planar surface, as in the embodiments shownand described in detail hereinafter.

As illustrated in a first embodiment depicted by FIG. 2(a), a firstsection of the lens is provided in the form of a hyper-hemisphere 100.The hyper-hemisphere 100 is generally made of fused silica in order tohave the same refractive index as the fused silica capillary. Thehyper-hemisphere in this illustrative embodiment has a diameter of 3 mm.

A substantially planar surface 102 is formed in the hyper-hemisphere100. The substantially planar surface is located at the internalaplanatic radius. As illustrated in FIG. 2(a), a curved groove 104 isformed along the diameter of the substantially planar surface of thehyper-hemisphere 100. The groove is a half cylinder sized to fit theoutside diameter of the capillary, for example, 360 μm.

A second section of the lens is configured to form a hemisphere 106. Thehemisphere 106 is made of fused silica to match the index of refractionof the capillary. Other transparent and non-fluorescing materials couldbe used for the hyper-hemisphere and hemisphere. However, optimalresults require that the index be close to that of the flowcell (orsimply, “cell”) or capillary. The hemisphere's dimensions in thisillustrative embodiment have been chosen so that the size of its planarsurface matches that of the hyper-hemisphere. The hemisphere could belarger or smaller than that illustrated without affecting performance.The hemispherical exterior surface 108 in the present embodiment isaluminized to form a retro-reflecting hemisphere that collectsforward-scattered or fluorescent light as shown in FIG. 2(a). Thehemisphere substantially increases the solid angle over which scatteredlight is collected. Additionally, the hemisphere increases the effectivepath length of the excitation beam through the sample leading to a verysubstantial increase in signal intensity.

A substantially planar surface 110 is formed on the hemisphere 106. Justas with that of the hyper-hemisphere, the substantially planar surface110 of the hemisphere 106 has a curved groove 112 formed along itsdiameter similar in dimensions to the groove in the hyper-hemisphere.

The substantially planar surfaces of the hyper-hemisphere and thehemisphere, 102 and 110 respectively, are mated such that the grooveforms a channel 114 as illustrated in FIG. 2(a). A fused silicacapillary 116 with an inside diameter of, for example, 50 μm, and anoutside diameter of, for example, 360 μm, is disposed in the channel.Narrow bore capillaries are typically used in this capillaryelectrophoresis embodiment because of heat transfer properties andconsiderations. As the bore size decreases, the surface area to volumeratio increases and, as a result, the heat transfer rate increases. Thisallows higher electric field strengths to be applied before Jouleheating begins to degrade performance. These higher electric fieldstrengths result in faster and more efficient separations. In addition,the lower electrical conductivity of the smaller solution volumes innarrow capillaries results in smaller currents and less Joule heatingfor a given applied field.

The air space 118 between the fused silica capillary and the groove isfilled with an index-matched liquid or gel such as a mineral oil, saltsolution, sugar solution, or the like. Index-matching liquids and gelscan be obtained commercially. It is important to choose one which istransparent and non-fluorescing at the wavelengths employed. Theindex-matched liquid or gel couples the lens with the capillary so thatthe two parts effectively form one optical component. Further, since thelens and capillary are made of the same material, the general lensingeffect of the capillary wall is eliminated.

A variation of the first embodiment can be constructed, as illustratedin FIG. 2(b), wherein constructing the curved grooves 104 and 112 iseliminated. Instead of forming curved grooves, the substantially planarsurfaces of both the hyper-hemisphere and hemisphere are ground backsuch that the fused silica capillary fits between them while maintainingthe optical alignment. The center of the capillary, or any optical pointof interest, is located at an aplanatic point. The air spaces 150 and152 on either side of the capillary are filled with index-matched gel orliquid (shown generally by label 154) so that, in essence, a singleoptical component is formed. This variation has all of the operatingcharacteristics and features contained within the above-describedembodiment and differs only in construction.

A second embodiment, illustrated in FIG. 3, of the present inventionutilizes only a single hyper-hemisphere with a substantially planarsurface. As with the first embodiment, the hyper-hemisphere is made offused silica in order to match the index of the capillary. Thesubstantially planar surface of the hyper-hemisphere is located in sucha manner so that the lumen of the capillary is at the internal aplanaticradius. The hyper-hemisphere may have a groove or not, analogous to thatdepicted in FIGS. 2a and 2 b, respectively. As illustrated in FIG. 3, afused silica capillary 116 is laid against the substantially planarsurface 102 of the hyper-hemisphere 100. Index-matching gel or liquid154 is placed around the capillary 116 so that the parts essentiallyform one optical component. The operating principle in this embodimentis similar to that governing the first embodiment except that thisembodiment does not use the enhancement factors including double-passingthe sample with excitation light and the absence of collecting theforward as well as back-scattered light.

FIG. 4 shows an optical system including the aplanatic capillaryinterface of the embodiment depicted in FIG. 3 used in a CE-Ramandetector, based around a Renishaw fibre probe. In this illustrativesystem, 514.5 nm light from an argon ion laser (not shown) is suppliedto the interface optics via a 50 μm optical fibre 400. The beam isreflected by a holographic notch filter 402 and focused by a microscopeobjective 404 into the hyper-hemisphere-capillary assembly 300. Thehyper-hemisphere provides an aberration-free focus of the laser beamwith the lumen of the capillary, as described above. For thisillustrative system, a microscope objective 404 with a working distancegreater than 3.7 mm is preferred. Back-scattered light from the sampleis collected via the microscope objective 404 and passed through notchfilters 406, 408 where the dominant Rayleigh scattered light is filteredout. The Raman scattered light is focused into an optical fibre 410 fortransmission to a spectrometer, for example, a Renishaw Mk 3spectrometer, which utilizes a cooled CCD array for detection. It willbe appreciated that this invention is equally applicable for both laserRaman as well as laser induced fluorescence.

Although the first embodiment described utilizes a retro-reflectinghemispherical surface to collect forward-scattered light, it should beappreciated that a mirror coating on the back of the capillary could beused to enhance the performance of the second embodiment. In this case,some portion of the laser light will double pass the sample volume ofinterest, thus a portion of the forward scattered or fluorescent lightwill be returned to the detection system.

Although the invention has been shown and described with respect toexemplary embodiments thereof, various other changes, omissions andadditions in the form and detail thereof may be made therein withoutdeparting from the spirit and scope of the invention.

What is claimed is:
 1. An optical apparatus for use in spectroscopy for analyzing samples, comprising: a single hyper-hemisphere, formed from a sphere having a substantially planar surface formed by truncating the circumference of said sphere whereby said single hyper-hemisphere has no protruding planar surface; and a cell, disposed upon said substantially planar surface of said hyper-hemisphere; wherein light passing through said hyper-hemisphere is focused aplanatically on said cell.
 2. The optical apparatus of claim 1, wherein said hyper-hemisphere and said cell are produced from material having substantially the same index.
 3. The optical apparatus of claim 1, wherein said cell is surrounded by a liquid.
 4. The optical apparatus of claim 3, wherein said liquid is indexed matched to said cell.
 5. The optical apparatus of claim 1 wherein said substantially planar surface of said hyper-hemisphere includes a curved groove, and said cell is disposed at least partially within said curved groove.
 6. The optical apparatus of claim 1 further including a reflecting coating on a portion of a surface of said cell.
 7. The optical apparatus of claim 1, wherein said cell is surrounded by a gel.
 8. The optical apparatus of claim 7, wherein said gel is indexed matched to said cell.
 9. The optical apparatus of claim 1, wherein said hyper-hemisphere and said cell are produced from fused silica.
 10. The optical apparatus of claim 1, wherein said cell includes a lumen and said cell is positioned so that said lumen of said cell is located at an internal aplanatic radius.
 11. A system for analyzing a sample using light spectroscopy, comprising: a light source; a microscopic objective, disposed to receive light from said light source; optical apparatus, disposed to receive light from said microscopic objective, said optical apparatus comprising: a single hyper-hemisphere, formed from a sphere having a substantially planar surface formed by truncating the circumference of said sphere whereby said single hyper-hemisphere has no protruding planar surface; a cell having a lumen, disposed proximate said substantially planar surface of said hyper-hemisphere, said cell disposed so that the lumen of the cell is at an internal aplanatic radius of said hyper hemisphere; whereby light passing through said hyper-hemisphere is focused aplanatically on said sample contained in said cell; and a light detection component, disposed to receive light that has passed through said sample.
 12. The apparatus of claim 11, wherein said light source is an argon ion laser.
 13. The apparatus of claim 11, further including an optical fiber whereby said light source is passed through said hyper-hemisphere via said optical fiber.
 14. The apparatus of claim 13, further including a holographic notch filter wherein said light source is passed through said hyper-hemisphere via said optical fiber by being reflected by said holographic notch filter.
 15. The apparatus of claim 14, further including a microscope objective wherein said light source is passed through said hyper-hemisphere via said optical fiber by being reflected by said holographic notch filter and focused by said microscope objective into said hyper-hemisphere. 